AFFINITY CHROMATOGRAPHY

Presented By:Salma Al-Salman

430204042

HISTORY• Affinity History 1930s, first developed by A .

Wilhelm Tiselius a Swedish biochemist , won the Nobel Prize in 1948

• Used to study enzymes and other proteins • Relies on the affinity of various biochemical

compounds with specific properties

Examples

• Antigen Antibody • Antibody Antigen • Substrate Enzyme • DNA Histon • Hormone Binding Protein/Receptor

Specificity of Affinity Chromatography

• Specificity is based on three aspect of affinity

1. Matrix : for ligand attachment.

2. Spacer arm : used to bind ligand to matrix

3. Ligand : molecule that binds reversibly to a specific target molecule(site of interaction)

Mechanism of Affinity Binding

Loading affinity column.

Proteins sieve through matrix of

affinity beads.

Proteins interact with affinity ligand with some binding loosely and others

tightly.

Wash off proteins that do

not bind.

Mechanism of Affinity Binding.. cont.

Wash off proteins that bind loosely.

Elute proteins that bind tightly to ligand and collect purified protein

of interest.

Mechanism of Affinity Binding.. cont.

• The Sample is injected into the equilibrated affinity chromatography column

• Only the substance with affinity for the ligand are retained on the column

• The substance with no affinity to the ligand will elute off

• The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent

Applications • Used in Genetic Engineering • Production of Vaccines

• And Basic Metabolic Research

ADVANTAGES OF AFFINITY CHROMATOGRAPHY

• Easy to achieve otherwise difficult separations • Often high purity in one step • Isolate pure target substance from excess amounts of contaminants• Remove specific contaminants • Fast separations

DISADVANTAGES OF AFFINITY CHROMATOGRAPHY

1) The interaction of proteins of interest and ligand has to be determined carefully.

2) This process required expensive materials, time, and small amount of protein that can be processed at once.

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